This Is AuburnElectronic Theses and Dissertations

Comprehensive Evaluation of Bacteroidales for Identification of Fecal Contamination Sources in Freshwater




Wijesinghe, Rasanthi

Type of Degree



Agronomy and Soils


Accurate identification of fecal contamination sources in an impaired watershed is crucial for developing best management practices. Microbial source tracking (MST) is commonly used to identify fecal pollution sources and includes both library-based and non-library-based approaches. The library-based approach often involves the development of a known source library from DNA fingerprints obtained from fecal samples of known host groups, allowing the DNA fingerprints from water samples to be compared against those in the known source library. The non-library based approach involves the direct detection of host-associated markers present in the water samples. In the first study, the library-based E. coli rep-PCR DNA fingerprinting method and Bacteroidales host-associated markers were used to identify fecal contamination sources in a mixed land-use watershed. DNA extracted from 64 water samples was analyzed using end-point and quantitative PCR. A total of 1,050 E. coli rep-PCR DNA fingerprints obtained from water samples were then compared against the known source library, which consisted of 945 unique E. coli DNA fingerprints from nine host groups. All of the water samples were positive for both general Bacteroidales markers and E. coli. The rep-PCR method detected human and cattle contamination in 94% and 75% of the water samples, respectively, while end-point PCR found human and cattle markers only in 27% and 28% of the samples. qPCR, on the other hand, detected human-associated markers in 64.0% of the samples. The second study evaluated human- and cattle-associated Bacteroidales genetic markers for their applicability in Alabama and identified the most suitable primer sets in qPCR assays for assessing fecal contamination in environmental samples. Four human and seven cattle-associated genetic markers were tested and human-associated HF183 and cattle-associated CowM3 appeared to be the best human and cattle markers, respectively. DNA extracted from surface water samples was amplified with general Bacteroidales primers as well as human- and cattle-associated primers. The results indicated that general Bacteroidales genetic markers were positive for all samples, with the highest concentration being 1,180,500 gene copies/100 ml. Human-associated Bacteroidales markers were detected in 87% of the water samples, while only 8% of the water samples contained the cattle-associated Bacteroidales markers. The major disadvantage of PCR is its inability to discriminate between DNA from live and dead cells. Propidium monoazide (PMA) is a DNA intercalating agent that can be used to detect DNA from live cells. The third study focused on the survival of E. coli and lives Bacteroidales in stream water and sediment microcosms. The general Bacteroidales markers were detectable up to 7 and 9 days in stream water and sediment microcosms, respectively, but human markers were detected only in the first 3 days in both microcosms. During the study period, a 3-log reduction of E. coli was observed, with 2,500 CFU/100 ml remaining in the water microcosm at the end of 14 days. However, E. coli survived in the sediment for more than 75 days. Experiments with PMA revealed that about 50% of the Bacteroidales gene copies amplified by qPCR were actually from dead cells or extracellular DNA. In summary, although both library-based and non-library-based MST methods can be used to detect the sources of fecal contamination in an impaired watershed, amplification of host associated Bacteroidales genetic markers with PMA-qPCR provides information on recent fecal pollution with a shorter turn-around time. More research should be devoted to developing viable cell-based approaches.